Quantification of Shiga toxin 2-encoding bacteriophages, by real-time PCR and correlation with phage infectivity

Aims: To evaluate a qPCR-based protocol for the enumeration of Shiga toxin (Stx) 2 phages and to compare the results of qPCR with the number of infective Stx phage particles. Methods and Results: An approach based on qPCR was applied to count Stx phages in five phage lysates of known titre. The number of viral particles from each phage lysate was determined by electron microscopy using latex spheres. The infectivity of the Stx phages was evaluated onto three bacterial host strains, by double agar layer assay and plaque blot hybridization. The number of phage particles detected by electron microscopy correlates with the number calculated by qPCR in all the phages assayed. The number of infectious phages was from 1 to 3 log(10) units below the numbers obtained by qPCR and electron microscopy. Conclusions: The approach allows accurate quantification of Stx phages with a high recovery. The number of infectious phages is always below the number of phage particles detected by qPCR. Significance and Impact of the Study: The qPCR method is a good approach to enumerate Stx phages. However, these results should be carefully considered when related to the number of infectious phages for each lysate that could be applied in real samples, because values of infectious particles are always below the number of Stx phages detected by qPCR.